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Alkaline Phosphatase Assay



  1. Buffer
    • 20 ul of dilution buffer and 160 ul of fluorescence buffer is needed for each sample
    • Prepare enough of the working buffer for the standards and unknowns
  2. Standards
    • Dilute stock control enzyme in water. 1:100 to 1:6400 has worked well. This correlates to 1 to 0.015625 ug/ml alkaline phosphatase.
  3. Samples
    • Dilute samples to ensure the samples fall within the standard curve. At least a 1:2 if not also a 1:10 dilution is recommended. Alternatively, you can aim for between 1 and 10 ug of protein per 20 ul.


  1. Prepare the wells by adding 20 ul of each standard or sample in triplicate.
  2. Add 180 ul of the working buffer to each well.
  3. Add 1 ul of the substrate to each well. Do this quickly as the substrate will immediately be converted.
  4. Measure fluorescence in the plate reader at 330 nm excitation and 440 nm. Generate a standard curve of FSU vs. alkaline phosphatase in ug/ml.